Investigations of the Ufm1 pathway and its association with a familial form of hip dysplasia A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2013 Michal Dudek School of Medicine Table of Contents Table of Contents .............................................................................................................. 2 List of Figures ................................................................................................................... 6 List of Tables..................................................................................................................... 9 List of Supplementary Figures ........................................................................................ 10 Abstract ........................................................................................................................... 11 Declaration ...................................................................................................................... 12 Copyright ........................................................................................................................ 13 Acknowledgements ......................................................................................................... 14 Abbreviations .................................................................................................................. 15 Chapter 1. Introduction ................................................................................................... 18 1.1 Chondrogenesis ..................................................................................................... 19 1.2 Joint development .................................................................................................. 20 1.3 Endochondral ossification ..................................................................................... 22 1.4 Articular cartilage .................................................................................................. 25 1.5. Bone formation ..................................................................................................... 29 1.5.1. Bone structure ................................................................................................ 29 1.5.2 Osteoblast differentiation ................................................................................ 30 1.5.3 Bone formation and remodelling .................................................................... 31 1.6 Skeletal disorders .................................................................................................. 32 1.6.1 Chondrodysplasias .......................................................................................... 32 1.6.2 Osteoarthritis ................................................................................................... 34 1.6.3 Hip dysplasia................................................................................................... 35 1.7 ER stress ................................................................................................................ 36 1.7.1 ER stress responses ......................................................................................... 36 1.7.2 Protein folding and quality control ................................................................. 37 1.7.3 ER stress and skeletal development ................................................................ 41 1.8 Ubiquitin-like pathways ........................................................................................ 44 1.8.1 The ubiquitin system ....................................................................................... 44 1.8.2 Ubiquitin-like proteins .................................................................................... 48 2 1.8.3 Ubiquitin and ubiquitin-like related disorders ................................................ 49 1.8.4 The Ufm1 system: a novel ubiquitin-like protein-conjugating system ........... 52 1.9 Background to the current study ........................................................................... 56 1.10 Hypothesis and aims of the study ........................................................................ 59 Chapter 2. Materials and Methods .................................................................................. 60 2.1 Materials ................................................................................................................ 60 2.2 Methods ................................................................................................................. 60 2.2.1 SDS-PAGE ..................................................................................................... 60 2.2.2 Western blotting .............................................................................................. 60 2.2.3 Cell culture ...................................................................................................... 60 2.2.4 Passaging ........................................................................................................ 61 2.2.5 Cell freezing/recover....................................................................................... 61 2.2.6 Calcium phosphate transfection ...................................................................... 61 2.2.7 Transfection using lipid based reagent ........................................................... 62 2.2.8 Lentiviral transduction .................................................................................... 62 2.2.9 Preparation of competent E. coli cells ............................................................ 62 2.2.10 Transformation of competent E. coli cells .................................................... 63 2.2.11 Polymerase chain reaction (PCR) ................................................................. 63 2.2.12 Agarose gel electrophoresis .......................................................................... 64 2.2.13 DNA extraction from an agarose gel ............................................................ 64 2.2.14 Plasmid DNA purification ............................................................................ 64 2.2.15 Mouse genomic DNA extraction .................................................................. 64 2.2.16 mRNA extraction .......................................................................................... 65 2.2.17 Generation of cDNA by reverse transcription PCR...................................... 65 2.2.18 Tandem affinity purification (TAP) .............................................................. 65 2.2.19 Radioactive in situ hybridisation .................................................................. 66 2.2.20 Osteogenic differentiation............................................................................. 68 2.2.21 Chondrogenic differentiation in micromass cultures .................................... 69 2.2.22 Qantitative real-time PCR ............................................................................. 69 2.2.23 Luciferase assay ............................................................................................ 69 2.2.24 Colloidal Blue staining ................................................................................. 70 2.2.25 Preparation of tissue sections for histology .................................................. 70 Chapter 3. Expression of the Ufm1 system ..................................................................... 72 3 3.1 Introduction ........................................................................................................... 72 3.2 Results ................................................................................................................... 73 3.2.1 Investigation of Ufsp2 gene expression in the hip joint during mouse development ............................................................................................................. 73 3.2.2 Expression of the Ufm1 system during osteogenic differentiation................. 78 3.2.3 Expression of the Ufm1 system during chondrogenic differentiation. ........... 82 3.2.4 Expression of the Ufm1 system in response to ER stress. .............................. 84 3.2.5 Analysis of the promoter regions of the Ufm1 system genes. ........................ 89 3.3 Discussion ............................................................................................................. 93 Chapter 4. Ufm1 biochemistry ........................................................................................ 99 4.1 Introduction ........................................................................................................... 99 4.2 Results ................................................................................................................. 100 4.2 Development of a purification method for isolation of Ufm1 conjugation targets. .................................................................................................................... 100 4.2.3 Large scale purification and mass spectrometry analysis of Ufm1 conjugated proteins................................................................................................................... 105 4.2.4 Testing of chemical agents potentially inducing conjugation of Ufm1 in the HEK293T cell line ................................................................................................. 109 4.2.5 Expression of the Ufm1 system in response to ER stress